Generation of haploid plants and improved plant breeding

ABSTRACT

Methods and compositions for generating haploid organisms are described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/088,065, filed Nov. 22, 2013, which is a continuation ofU.S. patent application Ser. No. 12/898,216, filed Oct. 5, 2010, nowU.S. Pat. No. 8,618,354, which claims benefit of priority to U.S.Provisional Patent Application No. 61/248,996, filed Oct. 6, 2009, eachof which is incorporated by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 081906-0961644_SEQ, created on Nov.11, 2015, (147,068 bytes, machine format IBM-PC, MS-Windows operatingsystem) is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Although plant breeding programs worldwide have made considerableprogress developing new cultivars with improved disease resistances,yields and other, useful traits, breeding as a whole relies on screeningnumerous plants to identify novel, desirable characteristics. Very largenumbers of progeny from crosses often must be grown and evaluated overseveral years in order to select one or a few plants with a desiredcombination of traits.

Standard breeding of diploid plants often requires screening andback-crossing of a large number of plants to achieve the desiredgenotype. One solution to the problem of screening large numbers ofprogeny has been to produce haploid plants, the chromosomes of which canbe doubled using colchicine or other means to achieve instantlyhomozygous, doubled-haploid plants.

Thus, marked improvements in the economics of breeding can be achievedvia doubled haploid production, since selection and other proceduralefficiencies can be markedly improved by using true-breeding(homozygous) progenies. With doubled haploid production systems,homozygosity is achieved in one generation. Thus, the breeder caneliminate the numerous cycles of inbreeding necessary by conventionalmethods to achieve practical levels of homozygosity. Indeed, truehomozygosity for all traits is not even achievable by conventionalbreeding methods.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for new ways for producing haploidorganisms.

In some embodiments, the invention provides a transgenic plantcomprising a heterologous transgene expression cassette, the expressioncassette comprising a promoter operably linked to a polynucleotideencoding a recombinantly altered CENH3, CENPC, MIS12, NDC80 or NUF2polypeptide, wherein in the event the recombinantly altered polypeptideis expressed in a first plant having a corresponding inactivatedendogenous CENH3, CENPC, MIS12, NDC80 or NUF2 gene and the first plantis crossed to a wildtype plant, at least 0.1% of resulting progeny arehaploid.

In some embodiments, one or two alleles of the endogenous CENH3, CENPC,MIS12, NDC80 or NUF2 genomic coding sequence of the plant is inactivatedor knocked out. In some embodiments, all alleles of the endogenousCENH3, CENPC, MIS12, NDC80 or NUF2 genomic coding sequence of the plantis inactivated or knocked out. In some embodiments, the plant, whencrossed with a wildtype plant, generates at least 0.1% (or, e.g., 0.5,1, 2, 5, 10, 20% or more) haploid progeny.

In some embodiments, the polypeptide is a recombinantly altered CENH3polypeptide. In some embodiments, the polypeptide comprises aheterologous amino acid sequence of at least 5 amino acids linked to aprotein comprising a CENH3 histone-fold domain, wherein the amino acidsequence is heterologous to the CENH3 histone-fold domain. In someembodiments, the heterologous amino acid sequence is linked directly tothe CENH3 histone-fold domain and the polypeptide lacks a CENH3 taildomain. In some embodiments, the heterologous amino acid sequence islinked to the CENH3 histone-fold domain via an intervening proteinsequence. In some embodiments, the intervening protein sequencecomprises a non-CENH3 histone H3 tail domain. In some embodiments, thenon-CENH3 histone H3 tail domain comprises an amino acid sequence atleast 70% identical to SEQ ID NO:95, or a fragment thereof at least 20amino acids long.

In some embodiments, the intervening protein sequence comprises a CENH3tail domain. In some embodiments, the intervening protein sequencecomprises a histone H3 tail domain and a heterologous histone CENH3 taildomain. In some embodiments, the CENH3 tail domain is heterologous tothe CENH3 histone-fold domain.

In some embodiments, the heterologous amino acid sequence is at least 10amino acids long. In some embodiments, the intervening protein sequencecomprises a histone H3 tail domain and a heterologous histone CENH3 taildomain. the heterologous amino acid sequence comprises green fluorescentprotein. In some embodiments, the heterologous amino acid sequencedisrupts centromeres. In some embodiments, the CENH3 histone-fold domainis selected from the group consisting of SEQ ID NOs: 49-94.

In some embodiments, the polypeptide comprises a non-CENH3 tail domainlinked to a CENH3 histone-fold domain.

In some embodiments, the polypeptide comprises a CENH3 histone-folddomain and a truncated CENH3 tail domain, wherein the amino terminus ofthe tail domain is truncated relative to the plant's endogenous taildomain. In some embodiments, the truncated CENH3 tail domain lacks threeor more amino terminal amino acids of the endogenous tail domain. Insome embodiments, a heterologous amino acid sequence is linked to theamino terminus of the truncated tail domain. In some embodiments, theheterologous amino acid sequence is at least 10 amino acids long. Insome embodiments, the heterologous amino acid sequence comprises greenfluorescent protein. In some embodiments, the heterologous amino acidsequence disrupts centromeres. In some embodiments, the CENH3histone-fold domain is selected from the group consisting of SEQ ID NOs:49-94.

In some embodiments, the polypeptide is a recombinantly altered CENPC,MIS12, NDC80 and NUF2 polypeptide.

The present invention also provides for an isolated nucleic acidcomprising a polynucleotide encoding a polypeptide, wherein thepolypeptide comprises:

a non-CENH3 tail domain linked to a CENH3 histone-fold domain; or

a truncated CENH3 tail domain linked to a CENH3 histone-fold domain,wherein the amino terminus of the tail domain is truncated.

The present invention also provides for a plant comprising a silencedCENH3 or one or two copies of an allele of a knocked out, inactivated,or mutated endogenous CENH3 gene.

The present invention also provides for method of generating a haploidplant, the method comprising,

crossing a plant expressing an endogenous CENH3 protein to a transgenicplant comprising a heterologous transgene expression cassette, theexpression cassette comprising a promoter operably linked to apolynucleotide encoding a recombinantly altered CENH3, CENPC, MIS12,NDC80 or NUF2 polypeptide, wherein in the event the recombinantlyaltered polypeptide is expressed in a first plant having a correspondinginactivated endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 gene and thefirst plant is crossed to a wildtype plant, at least 0.1% of resultingprogeny are haploid; andselecting F1 haploid progeny generated from the crossing step.

In some embodiments, the plant expressing an endogenous CENH3 protein isthe pollen parent of the cross.

In some embodiments, the plant expressing an endogenous CENH3 protein isthe ovule parent of the cross.

In some embodiments, the method further comprises converting at leastone selected haploid plant into a doubled haploid plant.

A method of making a transgenic plant comprising a heterologoustransgene expression cassette, the expression cassette comprising apromoter operably linked to a polynucleotide encoding a recombinantlyaltered CENH3, CENPC, MIS12, NDC80 or NUF2 polypeptide, wherein in theevent the recombinantly altered polypeptide is expressed in a firstplant having a corresponding inactivated endogenous CENH3, CENPC, MIS12,NDC80 or NUF2 gene and the first plant is crossed to a wildtype plant,at least 0.1% of resulting progeny are haploid, the method comprising,

transforming plant cells with a nucleic acid comprising the expressioncassette; and

selecting transformants comprising the nucleic acid, thereby making theplant.

In some embodiments, the present invention provides an isolatedpolynucleotide encoding a polypeptide, wherein the polypeptidecomprises:

an amino acid sequence of at least 5 amino acids linked to a proteincomprising a CENH3 histone-fold domain, wherein the amino acid sequenceis heterologous to the CENH3 histone-fold domain; or

a protein comprising a CENH3 histone-fold domain and a truncated CENH3tail domain, wherein the amino terminus of the tail domain is truncated.

In some embodiments, the heterologous amino acid sequence is linkeddirectly to the CENH3 histone-fold domain. In some embodiments, thepolypeptide lacks a CENH3 tail domain.

In some embodiments, the heterologous amino acid sequence is linked tothe CENH3 histone-fold domain via an intervening protein sequence. Insome embodiments, the intervening protein sequence comprises a non-CENH3histone H3 tail domain. In some embodiments, the intervening proteinsequence comprises a CENH3 tail domain. In some embodiments, the CENH3tail domain is heterologous to the CENH3 histone-fold domain. In someembodiments, the non-CENH3 histone H3 tail domain comprises an aminoacid sequence at least 70% identical to SEQ ID NO:95, or a fragmentthereof at least 20 amino acids long. In some embodiments, theintervening protein sequence comprises a histone H3 tail domain and aheterologous histone CENH3 tail domain.

In some embodiments, the heterologous amino acid sequence is at least 3,5, 10, 15, 20, 30, or 50 amino acids long, optionally lacking a fixedsecondary structure.

In some embodiments, the heterologous amino acid sequence comprisesgreen fluorescent protein.

In some embodiments, the heterologous amino acid sequence disruptscentromeres.

In some embodiments, the CENH3 histone-fold domain is selected from thegroup consisting of SEQ ID NOs: 49-94.

In some embodiments, the polypeptide comprises a protein comprising aCENH3 histone-fold domain and a truncated CENH3 tail domain, wherein theamino terminus of the tail domain is truncated.

In some embodiments, the truncated CENH3 tail domain lacks at least 1,2, 3, 4, 5, 6, 10, 15, or 20 amino terminal amino acids of theendogenous tail domain. In some embodiments, a heterologous amino acidsequence is linked to the amino terminus of the truncated tail domain.In some embodiments, the heterologous amino acid sequence is at least atleast 3, 5, 10, 15, 20, 30, or 50 amino acids long. In some embodiments,the heterologous amino acid sequence comprises green fluorescentprotein. In some embodiments, the heterologous amino acid sequencedisrupts centromeres.

In some embodiments, the CENH3 histone-fold domain is selected from thegroup consisting of SEQ ID NOs: 49-94.

The present invention also provides an expression cassette comprisingany of the above-listed the polynucleotides, wherein the expressioncassette comprises a promoter operably linked to the polynucleotideencoding a polypeptide. In some embodiments, the invention provides fora vector comprising the expression cassette.

In some embodiments, the invention provides for a plant comprising theexpression cassette.

In some embodiments, the heterologous histone tail domain comprises ahistone H3 tail domain or a heterologous histone CENH3 tail domain.

In some embodiments, the polypeptide comprises a histone H3 tail domainand a histone CENH3 tail domain.

In some embodiments, the plant comprises a silenced CENH3 or one or twocopies of an allele of a knocked out or mutated endogenous CENH3 gene.

In some embodiments, the expression cassette is integrated into thechromosome of the plant.

The present invention also provides for a plant comprising a silencedCENH3 or one or two copies of an allele of a knocked out or mutatedendogenous CENH3 gene.

The present invention also provides for a method of generating a haploidplant. In some embodiments, the method comprises, crossing a plantexpressing an endogenous CENH3 protein to the plant as described herein(e.g., expressing a tailswap protein); and

selecting F1 haploid progeny generated from the crossing step.

In some embodiments, the plant expressing an endogenous CENH3 protein isthe pollen parent of the cross.

In some embodiments, the plant expressing an endogenous CENH3 protein isthe ovule parent of the cross.

In some embodiments, the method further comprises converting at leastone selected haploid plant into a doubled haploid plant.

Other aspects of the invention will be clear from the remainder of thetext herein.

Definitions

An “endogenous” gene or protein sequence refers to a non-recombinantsequence of an organism as the sequence occurs in the organism beforehuman-induced mutation of the sequence. A “mutated” sequence refers to ahuman-altered sequence. Examples of human-induced mutation includeexposure of an organism to a high dose of chemical, radiological, orinsertional mutagen for the purposes of selecting mutants, as well asrecombinant alteration of a sequence. Examples of human-inducedrecombinant alterations can include, e.g., fusions, insertions,deletions, and/or changes to the sequence.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription and which are involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells. A plant promoter can be, butdoes not have to be, a nucleic acid sequence originally isolated from aplant.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like), and progeny of same. The class of plantsthat can be used in the method of the invention is generally as broad asthe class of higher and lower plants amenable to transformationtechniques, including angiosperms (monocotyledonous and dicotyledonousplants), gymnosperms, ferns, and multicellular algae. It includes plantsof a variety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous.

A polynucleotide or polypeptide sequence is “heterologous to” anorganism or a second sequence if it originates from a foreign species,or, if from the same species, is modified from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is not naturally associated with the promoter (e.g. agenetically engineered coding sequence or an allele from a differentecotype or variety). In another example, a CENH3 tail domain from afirst species is heterologous to a CENH3 histone-fold domain from asecond species.

“Recombinant” refers to a human manipulated polynucleotide or a copy orcomplement of a human manipulated polynucleotide. For instance, arecombinant expression cassette comprising a promoter operably linked toa second polynucleotide may include a promoter that is heterologous tothe second polynucleotide as the result of human manipulation (e.g., bymethods described in Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)or Current Protocols in Molecular Biology, Volumes 1-3, John Wiley &Sons, Inc. (1994-1998)). In another example, a recombinant expressioncassette may comprise polynucleotides combined in such a way that thepolynucleotides are extremely unlikely to be found in nature. Forinstance, human manipulated restriction sites or plasmid vectorsequences may flank or separate the promoter from the secondpolynucleotide. One of skill will recognize that polynucleotides can bemanipulated in many ways and are not limited to the examples above.

A “transgene” is used as the term is understood in the art and refers toa heterologous nucleic acid introduced into a cell by human molecularmanipulation of the cell's genome (e.g., by molecular transformation).Thus a “transgenic plant” is a plant comprising a transgene, i.e., is agenetically-modified plant. The transgenic plant can be the initialplant into which the transgene was introduced as well as progeny thereofwhose genome contain the transgene.

The term “corresponding” as used herein is used to mean “respective.”For example, where it is said that a plant contains a recombinantlyaltered copy of a protein selected from A, B, and C, and the plant alsocontains a “corresponding” mutated endogenous copy of the gene selectedfrom a gene encoding A, B, or C, if the plant contains a recombinantlyaltered protein A, the corresponding mutated endogenous copy would alsobe A. Alternatively, if the plant contains a recombinantly alteredprotein B, the corresponding mutated endogenous copy would also be B,etc.

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids mayalso include modified nucleotides that permit correct read through by apolymerase, and/or formation of double-stranded duplexes, and do notsignificantly alter expression of a polypeptide encoded by that nucleicacid.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidwhich directs the expression of a specific protein or peptide. Thenucleic acid sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The nucleic acid sequences include both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length sequences. It should be further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The phrase “host cell” refers to a cell from any organism. Exemplaryhost cells are derived from plants, bacteria, yeast, fungi, insects orother animals. Methods for introducing polynucleotide sequences intovarious types of host cells are well known in the art.

An “expression cassette” refers to a nucleic acid construct, which whenintroduced into a host cell (e.g., a plant cell), results intranscription and/or translation of a RNA or polypeptide, respectively.An expression cassette can result in transcription without translation,for example, when an siRNA or other non-protein encoding RNA istranscribed.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable on the Web through the National Center for BiotechnologyInformation (at ncbi.nlm.nih.gov). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity to a designated reference sequence. Alternatively, percentidentity can be any integer from 25% to 100%, for example, at least:25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% compared to a reference sequence using the programsdescribed herein; preferably BLAST using standard parameters, asdescribed below. One of skill will recognize that the percent identityvalues above can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of at least 40%.Percent identity of polypeptides can be any integer from 40% to 100%,for example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99%. In some embodiments, polypeptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Exemplary conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a sequence alignment of various CENH3 proteins (A.thaliana H3.3=SEQ ID NO:1; Human H3.3=SEQ ID NO:2; C. albicans=SEQ IDNO:106; Human=SEQ ID NO107; A. thaliana=SEQ ID NO:10; Poplar=SEQ IDNO:11; Rice=SEQ ID NO:108).

DETAILED DESCRIPTION

I. Introduction

The present invention is based, in part, on the surprising discoverythat elimination of an endogenous CENH3 in combination with expressionof a heterologous protein comprising an altered CENH3 results in a plantthat has useful properties for breeding. For example, when a plant thatlacks an endogenous CENH3 protein, and expresses a protein comprising(listed from amino terminus to carboxyl terminus) a GFP tag, a non-CENH3tail domain and a CENH3 histone fold domain, is crossed to a planthaving an endogenous CENH3 protein, a portion of the resulting progenylack all chromosomes derived from the parent plant that expresses analtered version of CENH3. Thus, the invention allows for the productionof haploid progeny. Haploid plants are useful, for example, forimproving and speeding breeding.

CENH3 is a member of the kinetochore complex, the protein structure onchromosomes where spindle fibers attach during cell division. Withoutintending to limit the scope of the invention, it is believed that theobserved results are due in part to generation of a kinetochore proteinthat acts more weakly than wildtype, thereby resulting in functionalkinetochore complexes (for example, in mitosis), but which result inrelatively poorly segregating chromosomes during meiosis relative tochromosomes also containing wildtype kinetochore complexes from theother parent. This results in functional kinetochore complexes when thealtered protein is the only isoform in the cell, but relatively poorlysegregating chromosomes during mitosis when the parent with alteredkinetochores is crossed to a parent with wildtype kinetochore complexes.In addition to CENH3, other kinetochore proteins include, e.g., CENPC,MCM21, MIS12, NDC80, and NUF2. Accordingly, the present inventionprovides for plants, fungi, or animals (or cells thereof) that express arecombinant mutated kinetochore protein (including but not limited toCENH3, CENPC, MCM21, MIS12, NDC80, and NUF2) that disrupts thecentromere, and/or plants, fungi, or animals (or cells thereof) in whichat least one or both copies of an allele of the endogenous CENH3 genehas been knocked out, mutated to reduce or eliminate its function, orsilenced. As explained in more detail below, the mutated kinetochoreprotein can be mutated in many different ways, including but not limitedto, as a “tailswap” protein, comprising a CENH3 histone-fold domain anda heterologous amino terminal sequence. The present invention alsoprovides for methods of generating a haploid plant by crossing a plantexpressing a mutated kinetochore protein (including but not limited to atailswap CENH3 protein), and not expressing an endogenous CENH3 protein,to a plant that expresses an endogenous CENH3 protein.

II. Kinetochore Proteins

A. CENH3 Proteins

CENH3 proteins are a well characterized class of proteins that arevariants of H3 histone proteins and that are specialized proteinsassociated with the centromere. CENH3 proteins are characterized by avariable tail domain, which does not form a rigid secondary structure,and a conserved histone fold domain made up of three α-helical regionsconnected by loop sections. Additional structural and functionalfeatures of CENH3 proteins can be found in, e.g., Cooper et al., MolBiol Evol. 21(9):1712-8 (2004); Malik et al., Nat Struct Biol.10(11):882-91 (2003); Black et al., Curr Opin Cell Biol. 20(1):91-100(2008). CENH3 proteins are one of the proteins that form the kinetochorecomplex.

A wide variety of CENH3 proteins have been identified. See, e.g., SEQ IDNOs:1-48. It will be appreciated that the above list is not intended tobe exhaustive and that additional CENH3 sequences are available fromgenomic studies or can be identified from genomic databases or bywell-known laboratory techniques. For example, where a particular plantor other organism species CENH3 is not readily available from adatabase, one can identify and clone the organism's CENH3 gene sequenceusing primers, which are optionally degenerate, based on conservedregions of other known CENH3 proteins.

The practice of the present invention will generally employ conventionalmethods of chemistry, biochemistry, molecular biology, cell biology,genetics, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds.(2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-HillCo.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press,Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook ofExperimental Immunology, Vols. I-IV, Blackwell Scientific Publications;Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual,2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4thedition, John Wiley & Sons; Ream et al., eds. (1998) Molecular BiologyTechniques: An Intensive Laboratory Course, Academic Press; Newton, C.R., and Graham, A., eds. (1997) PCR (Introduction to BiotechniquesSeries), 2nd ed., Springer Verlag.

i. CENH3 Histone Fold Domain

As noted above, the CENH3 histone fold domain is conserved between CENH3proteins from different species. The CENH3 histone fold domain can bedistinguished by three α-helical regions connected by loop sections.While it will be appreciated that the exact location of the histone folddomain will vary in CENH3 proteins from other species, it will be foundat the carboxyl terminus of an endogenous (wildtype) CENH3 protein.Thus, in some embodiments, a CENH3 protein can be identified in anendogenous protein as having a carboxyl terminal domain substantiallysimilar (e.g., at least 30%, 40%, 50%, 60%, 70%, 85%, 90%, 95% or moreidentity) to any of SEQ ID NO:s 49-94. An alignment of a selection ofCENH3 proteins is provided in FIG. 1 and illustrates areas ofconservation in the histone fold domain.

The border between the tail domain and the histone fold domain of CENH3proteins is at, within, or near (i.e., within 5, 10, 15, 20, or 25 aminoacids from the “P” of) the conserved PGTVAL (SEQ ID NO:114) sequence.The PGTVAL (SEQ ID NO:114) sequence is approximately 81 amino acids fromthe N terminus of the Arabidopsis CENH3 protein, though the distancefrom the N terminus of different endogenous CENH3 proteins varies. See,for example, the sequence listing. Thus, in some embodiments, thehistone fold region of CENH3 employed in the tailswap proteins includesall of the C-terminal amino acids of an endogenous CENH3 protein (or aprotein substantially similar to the endogenous sequence) up to andincluding the PGTVAL (SEQ ID NO:114). SEQ ID NOS:49-94 reflect thisoption. In other embodiments, the tailswap proteins of the invention cancomprise more or less of the CENH3 sequence. For example, in someembodiments, the tailswap will comprise the C-terminal sequence of aCENH3 protein, but only up to an amino acid 5, 10, 15, 20, or 25 aminoacids in the C-terminal direction from the “P” of the conserved PGTVAL(SEQ ID NO:114) sequence. In some embodiments, the tailswap willcomprise the C-terminal sequence of a CENH3 protein, but only up to anamino acid 5, 10, 15, 20, or 25 amino acids in the N-terminal directionfrom the “P” of the conserved PGTVAL (SEQ ID NO:114) sequence.

ii. CENH3 Histone Tail Domain

Although the histone-fold domain of CENH3 evolves more rapidly than thatof conventional H3, CENH3 and H3 histone-fold domains can still bealigned. In contrast, N-terminal tail domains of CENH3 are highlyvariable even between closely related species. Histone tail domains(including CENH3 tail domains) are flexible and unstructured, as shownby their lack of strong electron density in the structure of thenucleosome determined by X-ray crystallography (Luger et al., Nature389(6648):251-60 (1997)).

iii. Mutated CENH3 Proteins

Any number of mutations of CENH3 can be introduced into a CENH3 proteinto generate a mutated (including but not limited to a recombinantlyaltered) CENH3 protein capable of generating haploid plants whenexpressed in a plant lacking, or having suppressed expression of, anendogenous CENH3 protein, and where the resulting transgenic plant iscrossed to a plant expressing a wildtype CENH3 protein. Active mutatedCENH3 proteins can be identified, for example, by random mutagenesis, bysingle or multiple amino acid targeted mutagenesis, by generation ofcomplete or partial protein domain deletions, by fusion withheterologous amino acid sequences, or by combinations thereof. “Active”mutant CENH3 proteins refer to proteins, which when expressed in a plantin which CENH3 is knocked out or inactivated, results in viable plants,which viable plants when crossed to a wildtype plant, produce haploidprogeny at a more than normal frequency (e.g., at least 0.1, 0.5, 1, 5,10, 20% or more). Mutated CENH3 proteins can be readily tested byrecombinant expression of the mutated CENH3 protein in a plant lackingendogenous CENH3 protein, crossing the transgenic plant (as a male orfemale, depending on fertility) to a plant expression wildtype CENH3protein, and then screening for the production of haploid progeny.

In some embodiments, the mutated CENH3 protein is identical to anendogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g.,1-2, 1-4, 1-7) amino acids. For example, in some embodiments, theendogenous wildtype protein from the plant is identical or substantiallyidentical to any of SEQ ID NOs: 1-48 and the mutated CENH3 proteindiffers from the endogenous CENH3 protein by 1, 2, 3, 4, 5, 6, 7, 8, ormore (e.g., 1-2, 1-4, 1-7) amino acids.

In some embodiments, the mutated CENH3 protein contains a CENH3histone-fold domain identical to the CENH3 histone-fold domain of anendogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g.,1-2, 1-4, 1-7) amino acids. For example, in some embodiments, theendogenous wildtype CENH3 histone-fold domain from the plant isidentical or substantially identical to any of SEQ ID NOs: 49-94 and themutated CENH3 protein contains a CENH3 histone-fold domain that differsfrom the endogenous CENH3 protein histone-fold domain by 1, 2, 3, 4, 5,6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids.

It is believed that active CENH3 mutants include, for example, proteinscomprising:

a heterologous amino acid sequence (including but not limited to GFP)linked to a CENH3 truncated or complete tail domain or non-CENH3 taildomain, either of which is linked to a CENH3 histone fold domain; or

a CENH3 truncated tail domain, a heterologous CENH3 tail domain, ornon-CENH3 tail domain, either of which is linked to a CENH3 histone folddomain.

In some embodiments, the mutated CENH3 protein comprises a fusion of anamino-terminal heterologous amino acid sequence to the histone-folddomain of a CENH3 protein. Generally, the histone fold domain will beidentical or at least substantially identical to the CENH3 proteinendogenous to the organism in which the mutated CENH3 protein will beexpressed. In some embodiments, the mutated CENH3 protein will include ahistone tail domain, which can be, for example, a non-CENH3 tail domain,or a CENH3 tail domain.

It is believed that a large number of different amino acid sequences,when linked to a protein comprising a CENH3 histone-fold domain and asequence that can function as or replace a histone tail domain, can beused according to the present invention. In some embodiments, theheterologous sequence is linked directly to the CENH3 histone-folddomain. In some embodiments, the heterologous sequence is linked is anintervening amino acid sequence to the CENH3 histone-fold domain. Insome embodiments, the intervening amino acid sequence is an intact ortruncated CENH3 tail domain. In some embodiments, the heterologous aminoacid sequence, in combination with the histone-fold domain, will besufficient to prevent the lethality associated with loss of endogenousCENH3, but will sufficiently disrupt centromeres to allow for productionof haploid progeny, as discussed herein. Thus, in some embodiments, theheterologous amino acid sequence will comprise a portion that is, ormimics the function of, a histone tail domain and optionally can alsocomprise a bulky amino acid sequence that disrupts centromere function.In some embodiments, at least a portion of the heterologous amino acidsequence of the mutated CENH3 protein comprises any amino acid sequenceof at least 10, 20, 30, 40, 50, e.g., 10-30, 10-50, 20-50, 30-60 aminoacids, optionally lacking a stable secondary structure (e.g., lackingcoils, helices, or beta-sheets). In some embodiments, the tail domainhas less than 90, 80, or 70% identity with the tail domain (e.g., theN-terminal 135 amino acids) of the CENH3 protein endogenous to theorganism in which the mutated CENH3 protein will be expressed. In someembodiments, the tail domain of the mutated CENH3 protein comprises thetail domain of a non-CENH3 histone protein, including but not limited toan H3 histone protein. In some embodiments, the tail domain of themutated CENH3 protein comprises the tail domain of a non-CENH3 histoneprotein endogenous to the organism in which the mutated CENH3 proteinwill be expressed. In some embodiments, the tail domain of the mutatedCENH3 protein comprises the tail domain of a homologous or orthologous(from a different plant species) CENH3 tail. For example, it has beenfound that GFP fused to a maize CENH3 tail domain linked to anArabidopsis CENH3 histone-fold domain is active.

As noted above, in some embodiments, the tail domain of an H3 histone(not to be confused with a CENH3 histone) is used as the tail domainportion of the mutated CENH3 protein (these embodiments are sometimesreferred to as “tailswap” proteins). Plant H3 tail domains are wellconserved in various organisms. For example, a common H3 tail domainfrom plants is SEQ ID NO:95. Thus, in some embodiments, the heterologoustail portion of the tailswap protein will comprise an amino acidsequence substantially identical (e.g., at least 70, 80, 90, 95, or 100%identical) to SEQ ID NO:95, or a fragment thereof at least 15, 20, 25,30, 35, or 40 amino acids long.

In some embodiments, the mutated CENH3 proteins of the invention willlack at least a portion (e.g., at least 5, 10, 15, 20, 25, 30, or moreamino acids) of the endogenous CENH3 N-terminal region, and thus, insome embodiments, will have a truncated CENH3 tail domain compared to awildtype endogenous CENH3 protein. Mutated CENH3 proteins may, or maynot, be linked to a heterologous sequence.

Optionally, the heterologous amino acid sequence can comprise, orfurther comprise, one or more amino acid sequences at the amino and/orcarboxyl terminus and/or linking the tail and histone fold domains. Forexample, in some embodiments, the mutated CENH3 protein (e.g., atailswap or other CENH3 mutated protein) comprises a heterologous aminoacid sequence linked to the amino end of the tail domain. In someembodiments, the heterologous sequence is linked to the amino terminusof an otherwise wildtype CENH3 protein, wherein the heterologoussequence interferes with centromere function. For example, it has beenfound, for example, that green fluorescent protein, when linked towildtype CENH3, sufficiently disrupts centromeres to allow forproduction of haploid progeny. It is believed that the heterologoussequence can be any sequence that disrupts the CENH3 protein's abilityto maintain centromere function. Thus, in some embodiments, theheterologous sequence comprises a an amino acid sequence of at least 5,10, 15, 20, 25, 30, 50, or more kD.

In some embodiments, the mutated CENH3 protein will comprise a proteindomain that acts as a detectable or selectable marker. For example, anexemplary selectable marker protein is fluorescent or an antibiotic orherbicide resistance gene product. Selectable or detectable proteindomains are useful for monitoring the presence or absence of the mutatedCENH3 protein in an organism.

B. Non-CENH3 Kinetochore Proteins

It is believed that other proteins that make up the kinetochore complexcan also be mutated and expressed in a plant that otherwise does notexpress the corresponding endogenous kinetochore complex protein toresult in a viable plant which, when crossed to a wildtype plant havinga wildtype kinetochore complex, generates haploid progeny at a certainfrequency (e.g., at least 0.1, 0.5, 1, 5, 10, 20,%, or more). Exemplarynon-CENH3 members of the kinetochore complex include, e.g., CENPC,MCM21, MIS12, NDC80, and NUF2.

Active mutated non-CENH3 kinetochore complex proteins (e.g., CENPC,MCM21, MIS12, NDC80, or NUF2) can be identified, for example, by randommutagenesis, single or multiple amino acid targeted mutagenesis, bygeneration or complete or partial protein domain deletions, by fusionwith heterologous amino acid sequences, or combinations thereof “Active”mutant non-CENH3 kinetochore complex proteins refer to proteins, whichwhen expressed in a plant in which the corresponding non-CENH3kinetochore complex protein is knocked out or inactivated, results inviable plants, which when crossed to a wildtype plant, produce haploidprogeny at a more than normal frequency (e.g., at least 1, 5, 10, 20% ormore). In some embodiments, active mutated CENPC, MCM21, MIS12, NDC80,or NUF2 polypeptides are substantially identical to SEQ ID NOs: 96, 97,98, 99, or 100, respectively. Mutated non-CENH3 kinetochore complexproteins (e.g., CENPC, MCM21, MIS12, NDC80, or NUF2) can be readilytested by recombinant expression of the mutated non-CENH3 kinetochorecomplex protein in a plant lacking endogenous non-CENH3 kinetochorecomplex protein, crossing the transgenic plant (as a male or female,depending on fertility) to a plant expressing a wildtype non-CENH3kinetochore complex protein, and then screening for the production ofhaploid progeny.

In some embodiments, the mutated non-CENH3 kinetochore complex proteinis identical to an endogenous non-CENH3 kinetochore complex protein butfor 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids.For example, in some embodiments, the endogenous wildtype protein fromthe plant is identical or substantially identical to any of SEQ ID NOs:96, 97, 98, 99, or 100 and the mutated non-CENH3 kinetochore complexprotein differs from the endogenous non-CENH3 kinetochore complexprotein by 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) aminoacids.

Optionally, the heterologous amino acid sequence can comprise one ormore amino acid sequences at the amino and/or carboxyl terminus and/orlinking the tail and histone fold domains. For example, in someembodiments, the mutated non-CENH3 kinetochore complex protein comprisesa heterologous amino acid sequence linked to an amino end of thenon-CENH3 kinetochore complex protein. The heterologous sequence can beany sequence. In some embodiments, the heterologous sequence is linkedto the amino terminus of an otherwise wildtype non-CENH3 kinetochorecomplex protein, wherein the heterologous sequence interferes withcentromere function. In some embodiments, the heterologous sequencecomprises a an amino acid sequence of at least 5, 10, 15, 20, 25, 30,50, or more kD.

In some embodiments, the mutated non-CENH3 kinetochore complex proteinwill comprise a protein domain that acts as a detectable or selectablemarker. For example, an exemplary selectable marker protein isfluorescent or an antibiotic or herbicide resistance gene product.Selectable or detectable protein domains are useful for monitoring thepresence or absence of the mutated non-CENH3 kinetochore complex proteinin an organism.

III. Generation of Organisms of the Invention

The present invention provides for organisms that do not express, orexpress at reduced levels (e.g., less than 90, 80, 70, 60, 50, 40, 30,20, or 10% of wildtype levels), an endogenous CENH3 protein or non-CENH3kinetochore complex protein and optionally that express a correspondingmutated CENH3 protein or non-CENH3 kinetochore complex protein.Generally, lack of a kinetochore complex protein is lethal, unless atleast partially complemented by a mutated kinetochore complex protein asdescribed herein. Without intending to limit the scope of the invention,it is believed that there are several ways to make an organism thatlacks, or has reduced expression of, an endogenous kinetochore complexprotein but that expresses a mutated version of that protein.

In some embodiments, one can generate a CENH3 mutation in an endogenousCENH3 (or non-CENH3 kinetochore complex protein) gene that reduces oreliminates CENH3 activity or expression, or generate a kinetochorecomplex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) geneknockout. In these embodiments, one can generate an organismheterozygous for the gene knockout or mutation and introduce anexpression cassette for expression of the heterologous correspondingmutated kinetochore complex protein into the organism. Progeny from theheterozygote can then be selected that are homozygous for the mutationor knockout but that comprise the recombinantly expressed heterologousmutated kinetochore complex protein. Accordingly, the invention providesplants, plant cells or other organisms in which one or both CENH3alleles are knocked out or mutated to significantly or essentiallycompletely lack CENH3 activity, i.e., sufficient to induce embryolethality without a complementary expression of a mutated kinetochorecomplex protein as described herein (e.g., a tailswap protein). Theinvention also provides plants, plant cells or other organisms in whichone or both alleles of a non-CENH3 kinetochore complex gene are knockedout or mutated to significantly or essentially completely lack thecorresponding non-CENH3 kinetochore complex protein activity, i.e.,sufficient to induce embryo lethality without a complementary expressionof a mutated kinetochore complex protein as described herein. In plantshaving more than a diploid set of chromosomes (e.g. tetraploids), allalleles can be inactivated, mutated, or knocked out.

Alternatively, one can introduce the expression cassette encoding amutated kinetochore complex protein (e.g., including but not limited to,a tailswap protein) into an organism with an intact set of kinetochorecomplex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2)alleles and then silence the endogenous kinetochore complex protein(e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) gene by any way knownin the art. As an example, an siRNA or microRNA can be introduced orexpressed in the organism that reduces or eliminates expression of theendogenous kinetochore complex protein (e.g., CENH3, CENPC, MCM21,MIS12, NDC80, or NUF2) protein.

Ideally, the silencing siRNA or other silencing agent is selected tosilence the endogenous kinetochore complex protein (e.g., CENH3, CENPC,MCM21, MIS12, NDC80, or NUF2) gene but does not substantially interferewith expression of the mutated kinetochore complex protein (e.g., atailswap protein). In situations where endogenous CENH3 is to beinactivated, this can be achieved, for example, by targeting the siRNAto the N-terminal tail coding section, or untranslated portions, or theCENH3 mRNA, depending on the structure of the mutated kinetochorecomplex protein. Alternatively, the mutated kinetochore complex proteintransgene can be designed with novel codon usage, such that it lackssequence homology with the endogenous kinetochore complex protein geneand with the silencing siRNA.

IV. Reduction or Elimination of Endogenous Kinetochore Complex ProteinExpression

A number of methods can be used to inhibit, mutate, or inactivateexpression of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21,MIS12, NDC80, or NUF2) in plants. For instance, antisense technology canbe conveniently used to inactivate gene expression. To accomplish this,a nucleic acid segment from the desired gene is cloned and operablylinked to a promoter such that the antisense strand of RNA will betranscribed. The expression cassette is then transformed into plants andthe antisense strand of RNA is produced. In plant cells, it has beensuggested that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the polypeptide of interest, see,e.g., Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988);Pnueli et al., The Plant Cell 6:175-186 (1994); and Hiatt et al., U.S.Pat. No. 4,801,340.

The antisense nucleic acid sequence transformed into plants will besubstantially identical to at least a portion of the endogenous gene orgenes to be repressed. The sequence, however, does not have to beperfectly identical to inhibit expression. Thus, an antisense or sensenucleic acid molecule encoding only a portion of a kinetochore complexprotein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2), or a portionof the kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12,NDC80, or NUF2) mRNA (including but not limited to untranslated portionsof the mRNA) can be useful for producing a plant in which kinetochorecomplex protein expression is suppressed. The vectors of the presentinvention are optionally designed such that the inhibitory effectapplies only to a kinetochore complex protein (e.g., CENH3, CENPC,MCM21, MIS12, NDC80, or NUF2) and does not affect expression of othergenes. In situations where endogenous CENH3 is to be inactivated, onemethod for achieving this goal is to target the antisense sequence toCENH3 sequences (e.g., tail or untranslated mRNA sequences) not found inother proteins within a family of genes exhibiting homology orsubstantial homology to the CENH3 gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. For example, a sequence of betweenabout 30 or 40 nucleotides can be used, and in some embodiments, aboutfull length nucleotides should be used, though a sequence of at leastabout 20, 50, 100, 200, or 500 nucleotides can be used.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21,MIS12, NDC80, or NUF2) genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs that arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense suppression (also known asco-suppression). Introduction of expression cassettes in which a nucleicacid is configured in the sense orientation with respect to the promoterhas been shown to be an effective means by which to block thetranscription of target genes. For an example of the use of this methodto modulate expression of endogenous genes see, Napoli et al., The PlantCell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496(1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S.Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65% to the target a kinetochore complex protein (e.g., CENH3, CENPC,MCM21, MIS12, NDC80, or NUF2) sequence, but a higher identity can exerta more effective repression of expression of the endogenous sequences.In some embodiments, sequences with substantially greater identity areused, e.g., at least about 80, at least about 95%, or 100% identity areused. As with antisense regulation, the effect can be designed andtested so as to not significantly affect expression of other proteinswithin a similar family of genes exhibiting homology or substantialhomology.

For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants that are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. In some embodiments, a sequence of the size ranges notedabove for antisense regulation is used, i.e., 30-40, or at least about20, 50, 100, 200, 500 or more nucleotides.

Endogenous gene expression may also be suppressed by means of RNAinterference (RNAi) (and indeed co-suppression can be considered a typeof RNAi), which uses a double-stranded RNA having a sequence identicalor similar to the sequence of the target gene. RNAi is the phenomenon inwhich when a double-stranded RNA having a sequence identical or similarto that of the target gene is introduced into a cell, the expressions ofboth the inserted exogenous gene and target endogenous gene aresuppressed. The double-stranded RNA may be formed from two separatecomplementary RNAs or may be a single RNA with internally complementarysequences that form a double-stranded RNA. Although complete details ofthe mechanism of RNAi are still unknown, it is considered that theintroduced double-stranded RNA is initially cleaved into smallfragments, which then serve as indexes of the target gene in somemanner, thereby degrading the target gene. RNAi is known to be alsoeffective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc.Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl.Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431(1998); Matthew, Comp Funct. Genom. 5: 240-244 (2004); Lu, et al.,Nucleic Acids Research 32(21):e171 (2004)). For example, to achievesuppression of the expression of a kinetochore complex protein (e.g.,CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) using RNAi, adouble-stranded RNA having the sequence of an mRNA encoding thekinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, orNUF2), or a substantially similar sequence thereof (including thoseengineered not to translate the protein) or fragment thereof, isintroduced into a plant or other organism of interest. The resultingplants/organisms can then be screened for a phenotype associated withthe target protein (optionally in the presence of expression of atailswap protein to avoid lethality) and/or by monitoring steady-stateRNA levels for transcripts encoding the protein. Although the genes usedfor RNAi need not be completely identical to the target gene, they maybe at least 70%, 80%, 90%, 95% or more identical to the target (e.g.,CENH3 sequences as described herein) gene sequence. See, e.g., U.S.,Patent Publication No. 2004/0029283 for an example of a non-identicalsiRNA sequence used to suppress gene expression. The constructs encodingan RNA molecule with a stem-loop structure that is unrelated to thetarget gene and that is positioned distally to a sequence specific forthe gene of interest may also be used to inhibit target gene expression.See, e.g., U.S. Patent Publication No. 2003/0221211.

The RNAi polynucleotides can encompass the full-length target RNA or maycorrespond to a fragment of the target RNA. In some cases, the fragmentwill have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000nucleotides corresponding to the target sequence. In addition, in someembodiments, these fragments are at least, e.g., 10, 15, 20, 50, 100,150, 200, or more nucleotides in length. In some cases, fragments foruse in RNAi will be at least substantially similar to regions of atarget protein that do not occur in other proteins in the organism ormay be selected to have as little similarity to other organismtranscripts as possible, e.g., selected by comparison to sequences inanalyzing publicly-available sequence databases.

Expression vectors that continually express siRNA in transiently- andstably-transfected have been engineered to express small hairpin RNAs,which get processed in vivo into siRNAs molecules capable of carryingout gene-specific silencing (Brummelkamp et al., Science 296:550-553(2002), and Paddison, et al., Genes & Dev. 16:948-958 (2002)).Post-transcriptional gene silencing by double-stranded RNA is discussedin further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001),Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395:854 (1998).

One of skill in the art will recognize that sense (including but notlimited to siRNA) or antisense transcript should be targeted tosequences with the most variance between family members where the goalis to target only one (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2)histone family member.

Yet another way to suppress expression of an endogenous plant gene is byrecombinant expression of a microRNA that suppresses a target (e.g., aCENH3, CENPC, MCM21, MIS12, NDC80, or NUF2 gene). Artificial microRNAsare single-stranded RNAs (e.g., between 18-25 mers, generally 21 mers),that are not normally found in plants and that are processed fromendogenous miRNA precursors. Their sequences are designed according tothe determinants of plant miRNA target selection, such that theartificial microRNA specifically silences its intended target gene(s)and are generally described in Schwab et al, The Plant Cell 18:1121-1133(2006) as well as the internet-based methods of designing such microRNAsas described therein. See also, US Patent Publication No. 2008/0313773.

Methods for introducing genetic mutations into plant genes and selectingplants with desired traits are well known and can be used to introducemutations or to knock out a kinetochore complex protein (e.g., CENH3,CENPC, MCM21, MIS12, NDC80, or NUF2). For instance, seeds or other plantmaterial can be treated with a mutagenic insertional polynucleotide(e.g., transposon, T-DNA, etc.) or chemical substance, according tostandard techniques. Such chemical substances include, but are notlimited to, the following: diethyl sulfate, ethylene imine, ethylmethanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizingradiation from sources such as, X-rays or gamma rays can be used. Plantshaving mutated a kinetochore complex protein (e.g., CENH3, CENPC, MCM21,MIS12, NDC80, or NUF2) can then be identified, for example, by phenotypeor by molecular techniques.

Modified protein chains can also be readily designed utilizing variousrecombinant DNA techniques well known to those skilled in the art anddescribed for instance, in Sambrook et al., supra. Hydroxylamine canalso be used to introduce single base mutations into the coding regionof the gene (Sikorski et al., Meth. Enzymol., 194:302-318 (1991)). Forexample, the chains can vary from the naturally occurring sequence atthe primary structure level by amino acid substitutions, additions,deletions, and the like. These modifications can be used in a number ofcombinations to produce the final modified protein chain.

Alternatively, homologous recombination can be used to induce targetedgene modifications or knockouts by specifically targeting the akinetochore complex protein gene (e.g., CENH3, CENPC, MCM21, MIS12,NDC80, or NUF2) gene in vivo (see, generally, Grewal and Klar, Genetics,146:1221-1238 (1997) and Xu et al., Genes Dev., 10:2411-2422 (1996)).Homologous recombination has been demonstrated in plants (Puchta et al.,Experientia, 50:277-284 (1994); Swoboda et al., EMBO J., 13:484-489(1994); Offringa et al., Proc. Natl. Acad. Sci. USA, 90:7346-7350(1993); and Kempin et al., Nature, 389:802-803 (1997)).

In applying homologous recombination technology to the genes of theinvention, mutations in selected portions of an kinetochore complexprotein gene sequences (including 5′ upstream, 3′ downstream, andintragenic regions) such as those disclosed here are made in vitro andthen introduced into the desired plant using standard techniques. Sincethe efficiency of homologous recombination is known to be dependent onthe vectors used, use of dicistronic gene targeting vectors as describedby Mountford et al., Proc. Natl. Acad. Sci. USA, 91:4303-4307 (1994);and Vaulont et al., Transgenic Res., 4:247-255 (1995) are convenientlyused to increase the efficiency of selecting for altered CENH3 geneexpression in transgenic plants. The mutated gene will interact with thetarget wild-type gene in such a way that homologous recombination andtargeted replacement of the wild-type gene will occur in transgenicplant cells, resulting in suppression of kinetochore complex proteinactivity.

V. Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature,e.g., Weising et al., Ann. Rev. Genet. 22:421-477 (1988). A DNA sequencecoding for the desired polypeptide, for example the tailswap proteinfusions as described herein and/or siRNA, antisense, or other silencingconstructs, will be combined with transcriptional and translationalinitiation regulatory sequences which will direct the transcription ofthe sequence from the gene in the intended tissues of the transformedplant.

For example, a plant promoter fragment may be employed which will directexpression of the gene in all tissues of a regenerated plant.Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters), organ (organ-specific promoters) or may be otherwise undermore precise environmental control (inducible promoters). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues, such as fruit,seeds, flowers, pistils, or anthers. Suitable promoters include thosefrom genes encoding storage proteins or the lipid body membrane protein,oleosin.

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention can also comprise, for example, a markergene that confers a selectable phenotype on plant cells. For example,the marker may encode biocide resistance, particularly antibioticresistance, such as resistance to kanamycin, G418, bleomycin,hygromycin, or herbicide resistance, such as resistance tochlorosulfuron or Basta.

Constitutive Promoters

A promoter, or an active fragment thereof, can be employed which willdirect expression of a nucleic acid encoding a fusion protein of theinvention, in all transformed cells or tissues, e.g. as those of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include those from viruses which infectplants, such as the cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region (see, e.g., Dagless, Arch. Virol. 142:183-191 (1997));the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens(see, e.g., Mengiste supra (1997); O'Grady, Plant Mol. Biol. 29:99-108)(1995)); the promoter of the tobacco mosaic virus; the promoter ofFigwort mosaic virus (see, e.g., Maiti, Transgenic Res. 6:143-156)(1997)); actin promoters, such as the Arabidopsis actin gene promoter(see, e.g., Huang, Plant Mol. Biol. 33:125-139 (1997)); alcoholdehydrogenase (Adh) gene promoters (see, e.g., Millar, Plant Mol. Biol.31:897-904 (1996)); ACT11 from Arabidopsis (Huang et al., Plant Mol.Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147,Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe et al., Plant Physiol. 104:1167-1176 (1994)), GPc1from maize (GenBank No. X15596, Martinez et al., J. Mol. Biol.208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath etal., Plant Mol. Biol. 33:97-112 (1997)), other transcription initiationregions from various plant genes known to those of skill. See alsoHoltorf, “Comparison of different constitutive and inducible promotersfor the overexpression of transgenes in Arabidopsis thaliana,” PlantMol. Biol. 29:637-646 (1995). Additional constitutive promoters include,e.g., the polyubiquitin gene promoters from Arabidopsis thaliana, UBQ3and UBQ10, (Norris et al., Plant Mol. Biol. 21:895 (1993)), are alsouseful for directing gene expression.

Inducible Promoters

One can optionally use an inducible promoter to control (1) expressionof an artificial micro RNA, siRNA, or other silencing polynucleotide,(2) and simultaneously turn on expression of the transgenic mutated(e.g., tailswap) protein, or (3) both (1) and (2). This would have theadvantage of having a normal plant (e.g. one that might have higherfertility) until induction, which would then create gametes ready forinducing haploids.

Tissue-Specific Promoters

An alternative is to down-regulate the endogenous protein (e.g. by genesilencing) in a specific tissue (e.g., at least in the maturegametophytes (either pollen or embryo sac)) and to replace it only inthis tissue with a specific promoter that drives expression of atailswap protein. In some embodiments, the same tissue-specific promoteris used to drive an artificial micro RNA, siRNA, or other silencingpolynucleotide and the rescuing tailswap-encoding transgene.

VI. Production of Transgenic Plants or Plant Cells

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using biolistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal., Embo J. 3:2717-2722 (1984). Electroporation techniques aredescribed in Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985).Biolistic transformation techniques are described in Klein et al.,Nature 327:70-73 (1987).

Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al., Science233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. USA80:4803 (1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as increased disease resistance compared to a control plant thatwas not transformed or transformed with an empty vector. Suchregeneration techniques rely on manipulation of certain phytohormones ina tissue culture growth medium, typically relying on a biocide and/orherbicide marker which has been introduced together with the desirednucleotide sequences. Plant regeneration from cultured protoplasts isdescribed in Evans et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, NewYork, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtainedfrom plant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee et al., Ann. Rev. of PlantPhys. 38:467-486 (1987).

The nucleic acids and encoded polypeptides of the invention can be usedto confer the characteristics described herein, including the ability togenerate haploid progeny, as described herein, on essentially any plant.Thus, the invention has use over a broad range of plants, includingdicots or monocots, including e.g., species from the genera Asparagus,Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis,Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus,Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon,Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panicum,Pennisetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio,Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.

VII. Methods of Improved Breeding

Crossing plants that lack an endogenous kinetochore complex protein andexpress an active mutated kinetochore complex protein as describedherein (e.g., a tailswap or other mutated CENH3 or non-CENH3 kinetochorecomplex protein) either as a pollen or ovule parent to a plant thatexpresses an endogenous kinetochore complex protein (e.g., CENH3, CENPC,MCM21, MIS12, NDC80, or NUF2 protein) will result in at least someprogeny (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 20% or more) that arehaploid and comprise only chromosomes from the plant that expresses thekinetochore complex protein. Thus, the present invention allows for thegeneration of haploid plants having all of its chromosomes from a plantof interest by crossing the plant of interest with a planttransgenically expressing the mutated kinetochore complex protein andcollecting the resulting haploid seed.

As noted above, the plant expressing an endogenous wildtype CENH3protein can be crossed as either the male or female parent. One uniqueaspect of the present invention is that it allows for generation of aplant (or other organism) having only a male parent's nuclearchromosomes and a female parent's cytoplasm with associated mitochondriaand plastids, when the tailswap parent is the male parent.

While plants lacking an endogenous CENH3 gene and expressing a mutatedCENH3 protein made up of GFP-histone H3 tail-CENH3 histone-fold domainhave limited male fertility, it has been found that plants lacking anendogenous CENH3 gene and expressing both a mutated CENH3 protein madeup of GFP-histone H3 tail-CENH3 histone-fold domain and GFP-wildtypeCENH3 results in plants with higher male fertility making themconvenient for use as a male, as well as female, parent in crossing. Ingeneral, the invention provides for expression of two or more differentmutated kinetochore complex proteins in a plant (e.g., a plant lackingexpression of the corresponding endogenous kinetochore complexprotein(s).

Once generated, haploid plants can be used for a variety of usefulendeavors, including but not limited to the generation of doubledhaploid plants, which comprise an exact duplicate copy of chromosomes.Such doubled haploid plants are of particular use to speed plantbreeding, for example. A wide variety of methods are known forgenerating doubled haploid organisms from haploid organisms.

Somatic haploid cells, haploid embryos, haploid seeds, or haploid plantsproduced from haploid seeds can be treated with a chromosome doublingagent. Homozygous double haploid plants can be regenerated from haploidcells by contacting the haploid cells, including but not limited tohaploid callus, with chromosome doubling agents, such as colchicine,anti-microtubule herbicides, or nitrous oxide to create homozygousdoubled haploid cells.

Methods of chromosome doubling are disclosed in, for example, U.S. Pat.Nos. 5,770,788; 7,135,615, and US Patent Publication No. 2004/0210959and 2005/0289673; Antoine-Michard, S. et al., Plant Cell, Tissue OrganCult., Cordrecht, the Netherlands, Kluwer Academic Publishers48(3):203-207 (1997); Kato, A., Maize Genetics Cooperation Newsletter1997, 36-37; and Wan, Y. et al., Trends Genetics 77: 889-892 (1989).Wan, Y. et al., Trends Genetics 81: 205-211 (1991), the disclosures ofwhich are incorporated herein by reference. Methods can involve, forexample, contacting the haploid cell with nitrous oxide,anti-microtubule herbicides, or colchicine. Optionally, the haploids canbe transformed with a heterologous gene of interest, if desired.

Double haploid plants can be further crossed to other plants to generateF1, F2, or subsequent generations of plants with desired traits.

VIII. Non Plant Organisms

It is believed that the invention is also functional in non-plantorganisms that do not have unmatched sex chromosomes. Those of skill inthe art can thus generate a mutated kinetochore complex protein(including but not limited to a tailswap protein) based on a particularorganism's kinetochore complex protein (e.g., CENH3, CENPC, MCM21,MIS12, NDC80, or NUF2) protein sequence and knockout the correspondingendogenous kinetochore complex protein gene as appropriate for thatorganisms. Exemplary non-plant organisms for which the invention isbelieved to be applicable include, but is not limited to, yeast andother fungi, as well as to animals that lack unmatched (e.g., XY) sex orother chromosomes for whom haploids are not viable.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Production of haploid plants that inherit chromosomes from only oneparent can greatly accelerate plant breeding (Dunwell, J. M., PlantBiotechnol J in press; Forster, B. P. et al., Trends Plant Sci 12:368-75(2007); Forster, B. P. & Thomas, W. T. B. in Plant Breeding Reviews (ed.Janick, J.) 57-88 (John Wiley & Sons, Inc., 2005)). Haploids generatedfrom a heterozygous individual and converted to diploid create instanthomozygous lines, bypassing generations of inbreeding. Two methods aregenerally used to produce haploids: First, cultured gametophyte cellsmay be regenerated into haploid plants (Guha, S. & Maheshwari, S. C.,Nature 204:497 (1964)), but many species and genotypes are recalcitrantto this process (Forster, B. P. et al., Trends Plant Sci 12:368-75(2007); Wedzony, M. et al. in Advances in Haploid Production in HigherPlants (eds. Touraev, A., Forster, B. P. & Jain, S. M.) 1-33 (Springer,2009)). Second, haploids can be induced from rare interspecific crosses,in which one parental genome is eliminated after fertilization (Bains,G. S. & Howard, H. W., Nature 166:795 (1950); Barclay, I. R., Nature256:410-411 (1975); Burk, L. G. et al., Science 206:585 (1979); Clausen,R. E. & Mann, M. C., Proc Natl Acad Sci USA 10:121-124 (1924); Hougas,H. W. & Peloquin, S. J., Nature 180:1209-1210 (1957); Kasha, K. J. &Kao, K. N., Nature 225:874-6 (1970)). The molecular basis for genomeelimination is not understood, but one theory posits that centromeresfrom the two parent species interact unequally with the mitotic spindle,causing selective chromosome loss (Bennett, M. D. et al., Chromosoma54:175-200 (1976); Finch, R. A., Chromosoma 88:386-393 (1983); Laurie,D. A. & Bennett, M. D., Genome 32:953-961 (1989)). Here it is shown thathaploid Arabidopsis thaliana can be easily generated through seeds bymanipulating a single centromere protein, the centromere-specifichistone CENH3/CENP-A. When cenh3 null mutants expressing altered CENH3proteins are crossed to wild type, chromosomes from the mutant areeliminated, producing haploid progeny. Haploids are spontaneouslyconverted into fertile diploids through meiotic non-reduction, allowingtheir genotype to be perpetuated. Maternal and paternal haploids can begenerated through reciprocal crosses. Centromere-mediated genomeelimination has also been exploited to convert a natural tetraploidArabidopsis into a diploid, reducing its ploidy to simplify breeding. AsCENH3 is universal in eukaryotes, our method can be extended to producehaploids in any plant species.

Centromeres are the chromosomal loci that attach to spindle microtubulesto mediate faithful inheritance of the genome during cell division. Theyare epigenetically specified by incorporation of CENH3 (CENP-A inhumans, HTR12 in A. thaliana (Talbert, P. B. et al., Plant Cell14:1053-66 (2002))), a histone H3 variant that replaces conventional H3in centromeric nucleosomes (Henikoff, S. & Dalal, Y., Curr Opin GenetDev 15:177-84 (2005))). Cenh3-1, an embryo-lethal null mutant in A.thaliana that allows us to completely replace native CENH3 with modifiedvariants, was isolated. cenh3-1 plants complemented by transgenic greenfluorescent protein-tagged CENH3 (GFP-CENH3) have a wild-type phenotype.cenh3-1 can also be rescued by “GFP-tailswap”, a transgene in which thehypervariable N-terminal tail domain of CENH3 was replaced with the tailof conventional H3, using the H3.3 variant (encoded by At1g13370).

GFP-tailswap was tagged at its N-terminus with GF and contained theN-terminal tail of H3 fused to the histone fold domain of CENH3 asfollows:

H3 tail: MARTKQSARKSHGGKAPTKQLATKAARKSAPTTGGVKKPHRFR (SEQ ID NO:95)joined to the CENH3 histone fold domain:PGTVALKEIRHFQKQTNLLIPAASFIREVRSITHMLAPPQINRWTAEALVALQEAAEDYLVGLFSDSMLCAIHARRVTLMRKDFELARRLGGKGRPW (SEQ ID NO:109).

“GFP-tailswap” plants (cenh3-1 rescued by a GFP-tailswap transgene)showed accurate mitosis, as aneuploidy in somatic cells was notdetected. However, GFP-tailswap plants were sterile upon flowering,indicating that they may have a specific defect in meiosis. GFP-tailswapwas mostly male sterile, although it could be used as a pollen donor ifmany anthers were pooled. When crossed as the female to a wild typemale, GFP-tailswap plants were 60-70% as fertile as wild type.

When GFP-tailswap was pollinated by wild type, several unusualphenotypes in F1 progeny were observed. First, 80-95% of fertilizedovules aborted early in development, yielding inviable seeds (Table 1).

TABLE 1 Haploid plants contain only the nuclear genome of their wildtype parent. seeds/ % normal total plants Cross silique seed analyzedhaploids diploids aneuploids (%) (%) (%) WT Col-0 × 52 ± 6 99.5 224 0(0) 224 (100) 0 (0) WT Col-0 (n = 23) GFP-tailswap × 0.6 80 213 0 (0)197 (92)  16 (8)  GFP-tailswap (n = 1206) GFP-tailswap × 32 ± 9 12 67 23(34) 23 (34) 21 (32) WT Col-0 (n = 40) WT Col-0 × nd nd 116 5 (4) 99(85) 12 (11) GFP-tailswap GFP-tailswap × 30 ± 4 23 127 32* (25) 32 (25)63 (50) WT Ler (n = 22) GFP-tailswap × 23 ± 5 8 22 10* (45)  7 (32)  5(28) WT Ws-0 (n = 14) GFP-tailswap × 28 ± 5 30 117 34† (29) 39 (33) 44(38) WT C24/Ler (n = 13) C24/Ler male- 22 ± 14 63 226 12† (5) 206 (91) 8 (4) sterile × (n = 18) GFP-tailswap GFP-CENH3 × 53 ± 4 99 209 0 (0)209 (100) 0 (0) GFP-CENH3 (n = 21) GFP-CENH3 × 54 ± 7 67 164 8 (5) 109(66)  47 (29) WT (n = 18) WT × 48 ± 6 96 112 0 (0) 108 (96)  4 (4)GFP-CENH3 (n = 13) diploids triploids aneuploids (%) (%) (%)GFP-tailswap × 21 ± 6 1.8 41 11 (27) 0 30 (73) Wa-1 (tetraploid) (n =96)

Second, while viable offspring were expected to be diploids heterozygousfor cenh3-1 and hemizygous for the GFP-tailswap transgene, 10 out of 16plants had only wild-type CENH3 and lacked GFP-tailswap. Each of theseplants was sterile despite having a wild-type genotype. Furthermore,crossing GFP-tailswap to a quartet mutant male also yielded sterile F1offspring (⅗ plants) that showed the quartet mutant phenotype of fusedpollen, despite the fact that quartet is recessive and the GFP-tailswapparent was expected to transmit a wild-type QUARTET allele. Thesestriking observations suggested that sterile progeny had lostchromosomes from their GFP-tailswap mother, and thus had fewerchromosomes than diploid A. thaliana (2n=10). The karyotype of theseplants was examined and found them to be haploids containing only fivechromosomes.

As centromeres control chromosome inheritance, it was reasoned thatchromosomes that entered the zygote containing the GFP-tailswap variantof CENH3 would be missegregated and lost, creating haploid plants withchromosomes only from their wild type parent. To confirm this,GFP-tailswap plants (in the Col-0 accession) were crossed to severalpolymorphic accessions and genotyped F1 haploids for markers on all fiveA. thaliana chromosomes (Table 1). Regardless of the wild-type parentused, haploid plants invariably contained only wild-type chromosomes(paternal haploids), indicating that the GFP-tailswap genome waseliminated (a total of 42 haploids were genotyped). Further, our resultsshow that the process of inducing haploids by centromere-mediated genomeelimination is independent of the genotype of the wild-type parent.

Genome elimination induced by CENH3 alterations is not specific to theGFP-tailswap transgene. Crossing cenh3-1 mutants complemented byGFP-CENH3 to wild type also yielded haploid plants, but at a lowerfrequency than GFP-tailswap (Table 1). Haploid progeny fromself-fertilized GFP-tailswap or GFP-CENH3 plants were not observed(Table 1). Our results suggest that general perturbations in centromerestructure are sufficient to impede chromosome segregation during zygoticmitosis, creating a haploid embryo when chromosomes containing mutantCENH3 compete with wild type on the same spindle.

Haploids are efficiently generated from a GFP-tailswap×wild type cross,comprising 25-45% of viable offspring (Table 1). Remaining progeny wereeither diploid hybrids, or aneuploid hybrids showing the developmentalphenotypes typical of A. thaliana plants with more than 10 chromosomes(Henry, I. M. et al., Genetics 170:1979-88 (2005)) (Table 1). Aneuploidymight also account for the high level of seed abortion in aGFP-tailswap×wild type cross, as some embryos with unbalanced karyotypesmay be inviable.

Uniparental haploids may contain the genome of either their female ormale parent. Haploids were also obtained by crossing a wild-type femaleto GFP-tailswap as the pollen donor (Table 1). In this case, haploidprogeny are purely maternal in origin. Genotyping of the plastid genomeshowed that both maternal and paternal haploids contained the cytoplasmof their maternal parent. Either maternal or paternal haploids were madeby using GFP-tailswap plants as the male or female parent respectivelyin a cross to wild type. The proportion of haploids and aneuploids wasmuch lower if a wild-type female was crossed to a GFP-tailswap male(Table 1). It is hypothesized that if CENH3 is expressed earlier indevelopment from the maternal (wild type) genome, wild-type CENH3 couldbe incorporated into paternal chromosomes derived from GFP-tailswap,preventing genome elimination in a wild type×GFP-tailswap cross.

Haploid A. thaliana plants are morphologically similar to diploids, butare comparatively smaller in size. Early in vegetative development,haploids have narrower rosette leaves. After bolting, haploids producemore leaves from secondary meristems. Haploid flowers are smaller thandiploid flowers, following the general trend that flower size increaseswith ploidy in A. thaliana. Haploids are generally sterile. They containa single copy of each chromosome and cannot undergo homologue pairing inmeiosis, resulting in gametes that do not contain a full complement ofchromosomes. Maternal and paternal haploid plants had similar adultmorphology. This is consistent with the fact that all documentedimprinting in A. thaliana occurs in the short-lived endosperm, astructure confined to the seed.

To exploit the potential of haploids in crop improvement, their genomeshould be doubled to generate fertile diploids (doubled haploids)(Forster, B. P., et al. Trends Plant Sci 12:368-75 (2007)). A closeinspection of A. thaliana haploids revealed that random siliques had oneor two seeds. Each haploid plant yielded a total of 50-2500 seedsdepending on the wild-type parental accession (Table S1).

TABLE S1 Number of spontaneous diploid seeds produced by A. thalianahaploids Plant Col-0 Ler Ws-0 1 54 951 1662 2 68 2115 293 3 91 352 23434 214 520 1532 5 349 325 2679 6 421 101 215 7 537 219 913 8 121 1013 9134 424 10 85 630 11 99 1346 Mean 197.5454545 726.9090909 1376.714286Standard deviation 164.1294389 594.8035734 955.1793399

A majority (95%) of these seeds appeared normal and gave rise to fertilediploids. To address how haploids gave rise to diploid seeds, chromosomesegregation during haploid male meiosis was analyzed. During prophase Ithe five chromosomes remained separate as univalents, which alignedproperly in metaphase I. In anaphase I, most meiocytes showed unbalancedreductional segregation (4-1, 3-2, etc.). Meiosis II in these cases gaverise to aneuploid tetrads. In a small minority of anaphase I cells, the5 univalents migrated towards one pole (5-0 segregation). In subsequentmeiosis II, sister chromatids segregated equally, giving rise to haploiddyads and viable gametes. Thus, it is assumed that occasionalnon-reduction during both male and female haploid meiosis yieldeddoubled haploids through self-fertilization, consistent with previousobservations (Chase, S. S., Botanical Review 35:117-167 (1969); Jauhar,P. P. et al., Crop Science 40:1742-1749 (2000)). In rare instances,spontaneous chromosome doubling in somatic tissues of haploid A.thaliana plants was observed; a side branch from the main inflorescence(2 out of 78 plants) or a random silique (6 out of 78 plants) showed acomplete seed set. The microtubule polymerization inhibitor colchicinealso induces somatic chromosome doubling in haploid A. thaliana, anddiploid shoots that regenerate after treatment show complete seed set.Although A. thaliana haploids have been produced through anther culture(Avetisov, V. A., Genetika, 12:17-25 (1976)), spontaneous diploidsrecovered in these experiments were reportedly sterile (Scholl, R. &Amos, J. A., Z Pflanzenphysiol 96:407-414 (1980)), and the method hasnot been widely adopted. The ease of generating haploids through seed byaltering CENH3, and of converting haploids into diploids allows largescale generation of doubled haploids in A. thaliana.

Many commercial crops are polyploid (Udall, J. A. & Wendel, J. F., CropSci, 46:S3-S14 (2006)), but genetic analysis of polyploids is tedious.Reducing the ploidy of these crops will facilitate easy breeding, so itwas tested whether centromere-mediated genome elimination could scaledown a tetraploid to diploid. A. thaliana is predominantly diploid, buttetraploid accessions exist (Henry, I. M. et al., Genetics 170:1979-88(2005)). GFP-tailswap was crossed to the natural tetraploid Warschau-1(Wa-1), and although over 98% of seed were aborted, viable F1 progenyincluded synthetic diploid plants containing only Wa-1 chromosomes(Table 1). Therefore, it is possible to extend centromere-mediatedgenome elimination to halve the ploidy of polyploids.

Centromere incompatibility was previously hypothesized to causeselective genome elimination in interspecies crosses (Bennett, M. D. etal., Chromosoma 54:175-200 (1976); Finch, R. A., Chromosoma 88:386-393(1983); Laurie, D. A. & Bennett, M. D., Genome 32:953-961 (1989);Heppich, S. et al., Theor Appl Genet 61:101-104 (1982); Jin, W. et al.,Plant Cell 16:571-81 (2004)), but it was not known how centromeres couldbe manipulated to achieve this. It was established a practical basis forengineering genome elimination by altering CENH3, a protein essentialfor centromere function in all eukaryotes. The fact that haploids wereproduced with both GFP-tailswap and GFP-CENH3 transgenes suggests thatmultiple different alterations to the protein may induce genomeelimination in other plants. A. thaliana plants that coexpress wild-typeand GFP-tailswap or GFP-CENH3 proteins do not act as a haploid inducer.Therefore, our method currently relies on replacing native CENH3 with analtered variant. A cenh3 mutation or a gene silencing method such as RNAinterference could be used to reduce or eliminate endogenous CENH3function in a novel species.

Haploid inducing lines have been described in the grasses (Coe, E. H.,American Naturalist 93:381-382 (1959); Hagberg, A. & Hagberg, G.,Hereditas 93:341-343 (1980); Kermicle, J. L., Science 166:1422-1424(1969)), but their genetic basis is not known, except for maizeindeterminate gametophyte (ig) (Evans, M. M., Plant Cell 19:46-62(2007)). The effect of ig may be limited to maize, because mutations inthe A. thaliana ig orthologue AS2 do not phenocopy its effect (Ori, N.et al., Development 127:5523-32 (2000)). Our process has key advantagesover current methods for producing haploid plants. 1) No tissue cultureis needed, removing a major source of genotype dependence. 2) The sameinducer produces maternal and paternal haploids. 3) Crossing a cenh3mutant as the female transfers the nuclear genome of the male parentinto a heterologous cytoplasm. This could accelerate production ofcytoplasmic male sterile lines for making hybrid seed. 4) Genomeelimination occurs between parents that are isogenic except for CENH3alterations, avoiding fertility barriers inherent to wide crosses.

Genome elimination induced by changes in CENH3 probably occurs duringthe first few zygotic mitoses, when centromeres from the two parents areloaded with different populations of CENH3 proteins. Expression of bothwild-type and mutant CENH3 genes in subsequent cell cycles shouldrapidly equalize the amount of the two proteins in individualcentromeres. Zygotic mitosis is normal in GFP-tailswap and in GFP-CENH3plants, because haploids from self-fertilized plants were not observed.Furthermore, GFP-CENH3 plants have a completely wild type phenotype.Subtle differences in centromere DNA binding, kinetochore assembly, orcoupling to spindle microtubules may be sufficient to slow thesegregation of chromosomes containing altered CENH3, resulting in genomeelimination. Cell cycle checkpoints in plants must be relaxed enough toallow wild type and mutant chromosomes to segregate differentially, andpresumably to permit cytokinesis without complete chromosomesegregation. The precise mechanism of genome elimination in ourexperiments remains unknown.

Centromere DNA sequences and the CENH3 protein both evolve rapidly, andcentromere differences have been proposed to create species barriers(Henikoff, S. et al., Science 293:1098-102 (2001)). Although ourexperiments used tagged proteins, they indicate that changes in CENH3can induce specific chromosome loss in a hybrid zygote.

Methods Summary

Plant Materials.

cenh3-1 is a G-to-A transition at nucleotide 161 relative to ATG=+1, andmutates a conserved splice acceptor in the second intron. GFP-CENH3 andGFP-tailswap transgenes contained an N-terminal GFP, and used theendogenous CENH3 promoter and terminator. The location of theGFP-tailswap transgene was determined by TAIL-PCR, allowing us todetermine whether the transgene was homozygous or hemizygous. TheC24/Ler male sterile line was a gift from Dr Luca Comai (University ofCalifornia, Davis). Male sterility was conferred by the A9-barnasetransgene. Plants were grown under 16 hrs of light/8 hours of dark at 20degrees C.

Genomic DNA Preparation and Genotyping.

Genomic DNA preparation and PCR genotyping were performed using standardmethods.

Cytogenetic Analysis.

To analyze meiotic progression and to determine ploidy, mitotic andmeiotic chromosome spreads from anthers were prepared according topublished protocols.

Plant Materials

cenh3-1 was isolated by the TILLING procedure (Comai, L. & Henikoff, S.,Plant J 45:684-94 (2006)). The TILLING population was created bymutagenizing Arabidopsis thaliana in the Col-0 accession withethylmethanesulfonate, using standard protocols. Cenh3-1 was isolated byTILLING using the CEL1 heteroduplex cleavage assay, with PCR primersspecific for the CENH3/HTR12 gene.

cenh3-1 is predicted to disrupt normal splicing of CENH3, because itmutates a conserved splice acceptor site at the beginning of the secondcoding exon. Translation of an mRNA containing the first coding exonspliced to an incorrect location within CENH3 is predicted to yield only18 correct amino acids. As the histone-fold domain of CENH3 begins atamino acid residue 82, it is believed that cenh3-1 is a null allele(this is supported by its embryo-lethal phenotype).

Cloning of the GFP-CENH3 and GFP-tailswap transgenes, and constructionof the complemented cenh3-1 GFP-CENH3 and cenh3-1 GFP-tailswap lines aredescribed elsewhere (Ravi, Comai, Sundaresan, Chan et al, manuscript inpreparation). Primer sequences and full details are available onrequest.

To cross wild type as the female to GFP-tailswap as the male, adissecting microscope was used to directly observe pollen deposition onthe stigma (GFP-tailswap is mostly male-sterile). The amount of viablepollen in individual flowers of GFP-tailswap varies. Flowers thatclearly showed higher amounts of pollen were selected, and pollinatedwith more than 60 anthers (10 GFP-tailswap flowers) per wild type stigmato achieve the seed set reported in Table 1. Using an optivisor(magnifying lens) and approximately 12 anthers (2 GFP-tailswap flowers)per wild type stigma, a much lower seed set per silique was obtained.

The percentage of normal seeds was determined by visual inspection usinga dissecting microscope.

Seed from GFP-tailswap×wild type crosses were sown on 1×MS platescontaining 1% sucrose to maximize germination efficiency, particularlyof seed that had an abnormal appearance. Late germinating seeds werefrequently haploid.

The quartet mutant used was qrt1-2 (Francis, K. E. et al., Plant Physiol142:1004-13 (2006)).

Male sterility in the C24/Ler line was conferred by the A9-barnasetransgene (Bushell, C. et al., Plant Cell 15:1430-42 (2003); Paul, W. etal., Plant Mol Biol 19:611-22 (1992)).

In the GFP-tailswap×Wa-1 experiment, progeny from the GFP-tailswap×Wa-1cross that contained only Wa-1 chromosomes were confirmed as diploidusing chromosome spreads. Plants that were heterozygous for somechromosomes (Col-0 and Wa-1 markers) and homozygous for otherchromosomes (Wa-1 markers only) were scored as aneuploid. Triploidoffspring (heterozygous for markers on all chromosomes) were not found.A subset of plants were further karyotyped by means of chromosomespreads to confirm aneuploidy.

Cytogenetic Analysis.

Mitotic and meiotic chromosome spreads from anthers were preparedaccording to published protocols (Ross, K. J. et al., Chromosome Res4:507-16 (1996)).

Colchicine Treatment

Colchicine treatment of developing haploid plants used a previouslypublished protocol with minor modifications (Josefsson, C. et al., CurrBiol 16:1322-8 (2006)). A solution of 0.25% colchicine, 0.2% Silwet wasprepared, and a 20 μL drop was placed on the meristem prior to bolting.Plants became transiently sick after colchicine treatment. Uponrecovery, fertile inflorescences appeared from secondary meristemsindicating successful chromosome doubling. Haploid plants can also betreated after bolting, although the rate of success is considerablylower.

Example 2 GFP-Maizetailswap Creates F1 Haploids in a Cross to Wild Type

A chimera was created in which the A. thaliana CENH3 tail from CENH3 isreplaced with the CENH3 tail domain (SEQ ID NO:102) from maize (Zeamays), thereby generating a fusion of the maize CENH3 tail and A.thaliana CENH3 histone-fold domain, and transformed the fusion intocenh3-1 heterozygotes. As expected, this GFP-maizetailswap protein wastargeted to kinetochores and rescued the embryo-lethal phenotype ofcenh3-1. Complemented plants were more sterile than GFP-tailswapcomplemented plants, but had limited fertility when used as the female.When cenh3-1 GFP-maizetailswap females were crossed to wild-type males,2 haploids, 3 diploids and 5 aneuploids were found among a total of 10F1 progeny.

mCherry-Tailswap Creates F1 Haploids in a Cross to Wild Type

A transgene was created in which the GFP tag in GFP-tailswap wasreplaced with an N-terminal mCherry tag (mCherry (SEQ ID NO:105) is amonomeric version of the red fluorescent protein DsRed). From N-terminusto C-terminus, this protein contains mCherry, the tail domain ofArabidopsis thaliana H3.3, and the histone fold domain of Arabidopsisthaliana CENH3. mCherry-tailswap transgenes were transformed intocenh3-1 heterozygotes. When complemented cenh3-1 mCherry-tailswap plantswere crossed as female to wild-type male, 1 haploid, 6 aneuploids and 4diploids were observed from 11 F1 progeny.

The mCherry-tailswap construct was made as a CP 169 vector pCAMBIA 1300with an HTR promoter. The insert included a Mlu I site followed by the Nterminal mcherry Sal I XbaI followed by HTR12 terminator. The H3Tailswap fragment was synthesized by overlapping PCR and digested withSalI+Xba I and cloned into CP169 to make the mcherrytailswap construct.

A Tailswap Transgene with No GFP Tag Complements a Cenh3-1 Mutation.Complemented Plants Create F1 Haploids in a Cross to Wild Type

A transgene was created in which the GFP tag in GFP-tailswap wasremoved. From N-terminus to C-terminus, this protein contains the taildomain of Arabidopsis thaliana H3.3, and the histone fold domain ofArabidopsis thaliana CENH3. tailswap transgenes were transformed intocenh3-1 heterozygotes. When complemented cenh3-1 tailswap plants werecrossed as female to wild-type male, 4 haploids, 27 aneuploids and 67diploids were observed from 95 F1 progeny.

Co-Expression of Different CENH3 Variants Creates Desirable Propertiesin a Genome Elimination Strain.

The previously described GFP-tailswap plant (cenh3-1 mutant plantsrescued by a GFP-tailswap transgene) is a very efficient haploidinducer, but is difficult to cross as the pollen donor because it ismostly male sterile. GFP-CENH3 (cenh3-1 mutant plants rescued by aGFP-CENH3 transgene) is a weaker haploid inducer but is much morefertile. It was found that co-expression of GFP-CENH3 and GFP-tailswapin cenh3-1 plants would produce more viable pollen than GFP-tailswap,yet still induce genome elimination when these plants were crossed towild-type diploid or tetraploids. Indeed, cenh3-1 carrying bothGFP-CENH3 and GFP-tailswap transgenes (GEM; Genome Elimination caused bya Mix of cenh3 variants) plants produced ample pollen for crosses,although pollen viability was still lower than wild-type.

Crossing GEM females to wild-type males yielded 2 F1 haploids from 50progeny. When wild-type females were crossed to GEM males, one haploidwas found from 104 progeny.

GEM plants are a major improvement over GFP-tailswap or GFP-CENH3 whenthe wild-type parent is a tetraploid that has diploid gametes. When GEMplants were crossed as male or female to tetraploid wild-type,chromosomes from the GEM parent were eliminated in a subset of F1progeny (Table 3). GEM is fertile as either male or female, and showsefficient genome elimination when crossed to a tetraploid parent withdiploid gametes.

TABLE 2 Crosses between GEM and diploid wild-type plants produce genomeelimination. Total Uniparental* plant diploid cross (♀ × ♂) analysedTriploid Aneuploid plants Wild type 4n × GEM 85 53 27  5 GEM × Wild type4n 84 12 57 15

TABLE 3 Crosses between GEM and tetraploid wild-type plants producegenome elimination. Total Uniparental* plant haploid cross (♀ × ♂)analysed Diploid Aneuploid plants Wild type 2n × GEM 104 62 18 1 GEM ×Wild type 2n  50 36 12 2Methods for GFP-Maizetailswap Construction

Maize tailswap CENH3 transgene was constructed by fusing in frame theMaize CENH3 N-terminal tail (corresponding to 1-61 aa) and ArabidopsisCENH3 histone fold domain (corresponding to 82-179 aa) by overlappingPCR. The maize N terminal tail domain (206 bp) was amplified from maizecDNA using the primer combinations CP 384(5′-NNNNgtcgacATGGCTCGAACCAAGCACCA-3′ (SEQ ID NO:110), SalI site isitalicized) and CP 572 (5′-CAACGGTTCCTGGCCTCCAGCGGTGGC-3′ (SEQ IDNO:111)). The Arabidopsis HFD (950 bp) was amplified from genomic DNAusing primer combinations CP 571 (5′-GCCACCGCTGGAGGCCAGGAACCGTTG-3′ (SEQID NO:112)) and CP 375 (5′-NNNNtctagaTCACCATGGTCTGCCTTTTCCTCC-3′ (SEQ IDNO:113), XbaI site is italicized). The resultant fragments were gelpurified and used as a template to fuse them in an overlapping PCR usingprimer combinations CP 384 and CP 375. The resultant 1.15 kb fragment iscloned as a SalI-XbaI fragment in a binary vector CP 93 (derived frompCAMBIA 1300). The vector CP 93 contains GFP coding sequence upstream inframe with SalI-XbaI site and its expression is controlled by the 5′ and3′ regulatory sequences of Arabidopsis CENH3 gene.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A plant comprising a mutated CENH3 gene, whereinthe mutated CENH3 gene encodes a mutant CENH3 polypeptide having a CENH3tail-domain and a CENH3 histone-fold domain, wherein the mutant CENH3polypeptide is identical to a wildtype CENH3 polypeptide but for 1-7amino acid substitutions that occur in the CENH3 histone-fold domain,wherein the plant comprising the mutated CENH3 gene, when crossed with awildtype diploid plant, generates haploid progeny.
 2. The plant of claim1, wherein the plant comprises one copy of an allele of the mutatedCENH3 gene.
 3. The plant of claim 1, wherein the plant comprises twocopies of an allele of the mutated CENH3 gene.
 4. The plant of claim 1,wherein the mutant CENH3 polypeptide is at least 95% identical to any ofSEQ ID NOs:49-94.
 5. The plant of claim 1, wherein the wildtype CENH3polypeptide comprises any of SEQ ID NOs: 49-94.
 6. A method ofgenerating an F1 progeny plant having half the ploidy of a parent plantexpressing an endogenous wildtype CENH3 protein, the method comprising,crossing the parent plant to the plant of claim 1; and selecting F1progeny generated from the crossing step having half the ploidy of theparent plant.
 7. The method of claim 6, wherein the parent plant is thepollen parent.
 8. The method of claim 6, wherein the parent plant is theovule parent.
 9. The method of claim 6, further comprises converting atleast one selected haploid plant into a doubled haploid plant.
 10. Themethod of claim 6, wherein the wildtype CENH3 polypeptide comprises anyof SEQ ID NOs: 49-94.
 11. The method of claim 6, wherein the mutantCENH3 polypeptide is at least 95% identical to any of SEQ ID NOs: 49-94.